Materials - Tamoxifen, 4-hydroxy-tamoxifen, Dimethylsulfoxide, 2’,7’-dichlorofluorescin diacetate, 5,5’-dithio-bis (2-nitrobenzoic acid), colchicine, valspodar (PSC-833) and Tween-20 (P9416) were all purchased from Sigma Aldrich. Lipofectamine 2000, the microBCA kit and the Pierce™ ECL Western Blotting Substrate Kit were all products of Thermo Fisher. Propidium iodide and the HRP-conjugated goat anti-mouse were purchased from Invitrogen, and the IncuCyte® is a product of Essen BioScience.
Tissue culture and cell growth assays - Drug-sensitive (AuxB1, MDA-MB-231), -resistant (CHORC5, CHORC5/Bcl-2, MDA-Doxo400), and P-gp-knockout (AuxB1ΔP−gp, CHORC5 ΔP−gp−A1, CHORC5 ΔP−gp−A3) cells were grown in α-minimal essential medium (α-MEM) or Dulbecco’s modified eagle medium, supplemented with 10% fetal bovine serum (FBS; Gibco, Life technologies) at 37°C in the presence of 5% CO2, without or with selective concentrations of colchicine or doxorubicin (5 µg/ml for CHORC5 and 400 nM for MDA-Doxo400 cells; Sigma-Aldrich, Ont., CA). For cell proliferation assays, drug sensitive and resistant cells were plated in 200 µl α-MEM containing 10% FBS in 48-well plates. Cells were incubated for 24hrs at 37oC prior to the addition of 200 µl media containing colchicine, tamoxifen, hydrogen peroxide or rotenone alone and in combinations (Sigma-Aldrich, Ont., CA). Cell colonies were allowed to proliferate for 7–8 days at 37oC without or with drugs prior to the addition of cell-staining dye, methylene blue (0.1–1% methylene blue in ethanol/H2O). The dye solution was removed, and plates were washed gently in cold water and air-dried. The dye was extracted from fixed and stained cells with 0.1% SDS/PBS and the absorbance quantified at 660 nm (Dynatech Laboratories, MR5000). The effects of drugs on cell proliferation were determined by comparing the absorbance of cells grown in the presence of drugs to solvent control without added drugs (100% cell survival). All graphs shown represent the mean ± SD of three independent experiments done in triplicate. To assess the combined effects of different drugs, and measure potential drug synergy on cell proliferation, the method of Chou-Talay was used 25. Briefly, clonogenic cell proliferation assays were set up as described above using tamoxifen, rotenone, tamoxifen together with varying non-toxic concentrations of rotenone (0.4 nM or 1.3 nM) and rotenone together with varying non-toxic concentrations of tamoxifen (0.15 µM or 0.4 µM). The measured IC50 values of tamoxifen or rotenone for AuxB1 or CHORC5 were plotted on a Cartesian plane, and a linear regression was produced using the two points. The IC50 value of tamoxifen together with 0.4 nM or 1.3 nM of rotenone and rotenone together with tamoxifen (0.15 µM or 0.4 µM), normalized comparatively using the solvent control were plotted on the resulting graph for each cell line. Synergy is predicted when the resulting IC50 values for the combined drugs fall below the line connecting the two IC50 points for each drug alone on the x- and y-axes.
CRISPR/Cas9 knockout of P-gp in CHO cell lines - eSpCas9(1.1) was a gift from Feng Zhang (Addgene plasmid #71814; http://n2t.net/addgene:71814 ;RRID :Addgene_71814). The following guide-RNA sequences were designed against TMD1 of Cricetulus griseus P-gp, forward 5’-CACCGCTTATAGTTGCCTACATTC-3’ and reverse 5’-AAACGAATGTAGGCAACTATAA- GC-3’ primers. The constructs were transformed into TOP10 cells, and empty plasmid or plasmid containing the guide-RNA was isolated and transiently transfected into the AuxB1 or CHORC5 cells using the Lipofectamine 2000 kit (Invitrogen). Populations of the transfected cells were grown, and knockout clones were isolated via serial dilution method. The absence of P-gp was verified by Western blot using P-gp-specific monoclonal antibody, C219 mAb 26.
Apoptosis assays - For annexin-V staining of apoptotic cells, drug sensitive and resistant cells (AuxB1 and CHORC5, respectively) were seeded in six-well plates at 1–2 x 105 per well and incubated for 24hrs prior to the addition of tamoxifen for 24hrs. Cells (1x106) were lifted and washed with cold PBS and resuspended in 100 µl binding buffer according to the manufacturer’s protocol (BD, FITC Annexin V Apoptosis Detection kit I). Briefly, apoptotic cells stained with the addition of Annexin-V-FITC solution (5 µl) and propidium iodide (5µl), allowed to incubate for 15 minutes in the dark, then diluted with 400 µl of binding buffer prior to analysis by flow cytometry (BD FACSDiva). Percent apoptosis was determined by measuring the relative fluorescence in drug treated versus control untreated cells. For Hoechst dye staining of apoptotic cells, AuxB1 and CHORC5 cells were incubated without and with 5 µM tamoxifen for 24hr. Hoechst 33258 dye (1 µg/mL) was added for 10-min at 37°C prior to observing cells under UV light to assess the percent of apoptotic cell. Photographs were taken at 2000X magnification (Nikon, Eclipse TE200, Quebec, Canada).
ROS measurements - AuxB1 and CHORC5 cells were seeded at a density of 50,000 cells/well in a clear-bottomed black-well plate and allowed to adhere for 24hrs, after which the media was removed, and cells incubated with 100 µM H2DCFDA for 45 minutes at 37oC. Wells were washed with sterile cold HBSS, followed by the addition of 100 µl of fluorobrite DMEM + 8% FBS to each well. Tamoxifen (1 µM and 5 µM) were added and allowed to incubate for another 24hrs, after which the fluorescence signals were measured at 485ex, 527em using in the H4 Synergy plate reader (BioTek Inc., USA). The fluorescence signals from cells treated with tamoxifen relative to solvent control were plotted using GraphPad Prism (GraphPad Software, version 8.0.1).
Measurement of total reduced thiols - Cells (AuxB1 and CHORC5) were seeded at 100,000 cells/well in 48-well plate and allowed to adhere for 24hrs, after which increasing doses of tamoxifen were added. Following another 48-hour incubation, the media was removed and 250 µl of RIPA buffer (50 mM Tris, 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS, pH 8) was added to each well. Plates were mixed for 20 minutes and 50 µl of each sample was removed for protein measurement using micro BCA protein assay (ThermoFisher Scientific Inc. USA). To the remaining samples, 50 µl of 50 mM DTNB (5,5-dithio-bis-(2-nitrobenzoic acid) or Ellman's Reagent) was added and incubated for 30 minutes. The tissue culture plates were scanned at 412nm on Synergy H4™ Hybrid Multi-Mode Microplate reader (Biotek Inc. USA). Analysis was done comparing the absorbance of each sample to the total amount of protein in the sample, yielding the relative amount of sulfhydro moieties compared to solvent control. The data were analyzed and the total amount of sulfhydro groups compared to their respective solvent control were plotted using GraphPad Prism (GraphPad Software, version 8.0.1).
Protein extraction and immuno-detection - Total cell lysates (20 µg) from drug-sensitive and -resistant cells were resolved on 6% SDS-PAGE and transferred onto PVDF membrane. The membrane was blocked in milk in phosphate buffered saline (PBS) at 5% (w/v) and probed with P-gp specific mAb (0.1 µg/ml of C219 mAb) or anti-Bcl-2 (0.5 µg/ml; BioLegend, San Diego, USA) in 5% milk/PBS, followed by several washes in PBS and incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5000 v/v, BioRad, Ont., CA). The reactive signals were visualized using Western Breeze Chemiluminescent Kit and captured using ECL-imager from Thermo-Fisher Scientific. Tubulin expression was detected on the same PVDF membrane using anti-α-tubulin specific monoclonal antibody (1 µg/ml Sigma-Aldrich, Ont., CA), followed by HRP-conjugated goat anti-mouse IgG (1:5000 (v/v), BioRad, Ont., CA).
In vitro isobologram analysis - The effects of tamoxifen alone and in combination with rotenone on the proliferation of drug-sensitive (AuxB1 and MDA-MD-231) and –resistant (CHORC5 and MDA-Doxo400) cells were assessed using the modified fixed-ratio isobologram analysis protocol. This method is usually adopted to measure the degree of chemo-sensitization of a given compound and to detect if the effect between two drugs is synergistic, additive, or antagonistic 27. A stock of tamoxifen and rotenone combination was prepared in fixed-concentration ratios. Drug-sensitive and resistant cells were incubated with the above drug combination for 72h, and a nonlinear regression curve was used to determine the IC50 values for each drug alone and in combination. Fractional inhibitory concentrations (FICs) were then calculated using the equations below, as previously described 28,29.
The isobologram curves were constructed by plotting FICtam vs FICrotenone. The drug combination effect was assessed from the graphs but also by calculation. A straight diagonal line or FICindex=1 indicates a clear additive effect between the two drugs, while a concave curve below the diagonal of the graph denotes a synergistic effect between the drugs. In addition, the synergy may be strong or moderate when the means of FICindex values are respectively below 0.5 or between 0.5 and 1. A convex curve above the diagonal indicates an antagonistic effect and demonstrates an absence of synergy with the FICindex> 4. An FICindex between 1 and 4.0 is defined as no interaction 30.
Statistical analysis - All graphs and statistics were performed using GraphPad prism version 6. Statistics represent the student t and one-way ANOVA test.